JP2002253268A - New mutant n-acetyl glutamate synthase and method for producing l-arginine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an N-acetyl glutamate synthase having a mutation resistant to the feedback inhibition and a high activity, and to provide an L-arginine- producing bacterium having the enzyme. SOLUTION: This method for producing L-arginine comprises the steps of culturing an escherichia having a DNA encoding a mutant N-acetyl glutamate synthase which has an amino acid sequence modified by replacing the amino acid sequence corresponding to the 15th to 19th of a wild-type N-acetyl glutamate synthase with an amino acid sequence selected from sequence numbers 1 to 4 (see Specification) and is released from the feedback inhibition by L- arginine, accumulating L-arginine in a medium, and collecting L-arginine from the medium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the microbial industry, and to a method for producing L-arginine and the use of a novel feedback-inhibiting mutant enzyme in an arginine biosynthesis system of an arginine-producing bacterium of Escherichia coli.

[0002]

2. Description of the Related Art Biosynthesis of glutamic acid to arginine in Escherichia coli cells is carried out by a continuous reaction starting from glutamic acid acetylation by N-acetylglutamic acid synthase (hereinafter also referred to as "NAGS"). This process is regulated through arginine repression of regulon transcription and feedback inhibition of NAGS (Cunin R., et al., Microbiol. Rev., 50,
314-352, 1986). Arginine increases expression of argA by 250
Inhibits NAGS activity (Ki = 0.02 mM) (Le
isinger T., Haas D., J. Biol. Chem., 250, 1690-169
3, 1975). To enhance arginine biosynthesis in Escherichia coli, a NAGS enzyme that is resistant to feedback inhibition (fbr) is required.

[0003] Feedback-resistant mutant enzymes can be obtained by spontaneous mutation or by chemical or site-specific mutation. Several argA fbr mutations have been isolated,
Has been studied. Serratia marcescens shows that when an argA fbr mutation is present on a chromosome, cells become unstable, with reduced activity or altered affinity for glutamate.
gA mutation occurs (Takagi T, etal., J. Biochem., 99,
357-364, 1986).

The fbr argA gene derived from five strains of Escherichia coli having NAGS of fbr was cloned, and each fb argA gene was cloned.
r In the NAGS strain, separate single nucleotide substitutions were found in the argA gene, and His at position 15 to tyr, Tyr at position 19 to Cys, 5
Ser from position 4 to Asn, Arg from position 58 to His, position 287 from Gl
Substitutions from y to Ser and from Gln at position 432 to Arg have been clarified (Rajagopal, BS et al., Appl. Envi.
ron. Microbiol., 1998, v.64. No. 5, p.1805-1811).

The fbr trait of an enzyme often results from the substitution of an amino acid residue at one or several positions by another amino acid. These substitutions lead to reduced enzyme activity. For example, 2 in serine acetyltransferase (SAT) (cysE gene) of Escherichia coli
Substitution of the native Met at position 56 by another amino acid residue often results in the fbr trait, but the mutant SAT protein is not maintained at the level of activity of the native SAT (Nakamuri
S. et al., AEM, 64 (5): 1607-11, 1998). Thus, a disadvantage of the mutant enzymes obtained by these methods is that the mutant enzymes have lower activity than the wild-type enzymes.

[0006]

SUMMARY OF THE INVENTION The present invention provides an enzyme having an important role in arginine biosynthesis in Escherichia coli, having a mutation resistant to feedback inhibition and having high activity. That
This is one of the issues.

[0007]

According to the present invention, argA
Multiple sets of mutant argA by complete randomization of gene fragments
A novel synthesis of the gene is proposed. A variant in which the level of activity close to that of the natural form is maintained by simultaneously substituting some amino acids in a fragment of the protein sequence that can localize the fbr mutation, thereby maintaining the three-dimensional structure of the enzyme more correctly. A protein can be obtained.

Thus, the present invention described below has been made. (1) 15 types of wild-type N-acetylglutamic acid synthase
Mutant N-acetylglutamic acid synthesis in which the amino acid sequence corresponding to positions 19 to 19 has an amino acid sequence substituted with any one of SEQ ID NOs: 1 to 4, and the feedback inhibition by L-arginine has been released enzyme. (2) The mutant N-acetylglutamate synthetase according to (1), wherein the wild-type N-acetylglutamate synthase is Escherichia coli N-acetylglutamate synthase. (3) In one or more positions other than positions 15 to 19, substitution or deletion, insertion or addition of one or several amino acids is included, and feedback inhibition by L-arginine is released (1) Or, the mutant N-acetylglutamic acid synthase of (2). (4) A DNA encoding the mutant N-acetylglutamate synthase according to any one of (1) to (3). (5) A bacterium belonging to the genus Escherichia, which is transformed with the DNA of (4) and has an ability to produce L-arginine. (6) culturing the Escherichia bacterium of (5) in a medium;
-Arginine is produced and accumulated in the medium, and L-
A method for producing L-arginine, comprising collecting arginine.

In the present specification, NAGS having the fbr mutation as described above is referred to as mutant NAGS,
NA is sometimes referred to as a mutant argA gene. NAGS not having the mutation may be referred to as wild-type NAGS.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

<1> Mutant NAGS and Mutant argA Gene The mutant NAGS of the present invention and the mutant argA gene encoding the same were obtained by mutagenesis using randomized fragments. To obtain a large number of mutations in the argA gene, a complete randomization of a 15 nucleotide fragment of the argA gene encoding a region of amino acid residues 15 to 19 of the protein sequence was performed. Fragment of a full randomized 15 nucleotides, 4 ¹⁵ i.e. causing different DNA sequences of about 10 ^19, it can encode a different amino acid residue of 20 ⁵ in the peptide of 5 mer. The probability that a stop codon does not enter the frame in these sequences corresponds to about 0.95 ⁵ i.e. 78%. Therefore,
Complete randomization of the argA gene fragment encoding a peptide of amino acid residues 15 to 19 should result in approximately 2.5 million different protein sequences with the diversity of this peptide in the NAGS structure.

Subsequently, by selecting and screening a recombinant strain having the mutant argA gene cloned in the expression vector, the wild-type
We have successfully selected fbr mutants with various levels of bioactivity up to the level of NAGS activity. At that time, argD ^-, and, proB ^- or proA ^- the strain, and NAGS when an excess of L- arginine in the medium is present is inhibited L
-Proline cannot be synthesized and cannot grow, but if it retains fbr NAGS, it can grow on minimal medium,
It was thought that the argA holding strain could be selected. But,
As shown in the Examples below, this method has high activity fbr
It is difficult to obtain NAGS, and it has been found that fbr NAGS with high activity can be obtained by introducing a mutant argA gene into a wild type strain and selecting a strain that grows slowly.

According to the present invention, the sequence of mutant NAGS suitable as NAGS of the fbr trait has been clarified.
And a mutant argA gene encoding the same can be prepared by introducing a mutation into the wild-type argA gene according to a conventional method based on the sequence. The wild-type argA gene includes Escherichia coli
argA gene (GenBank Accession Y00492). The argA gene of Escherichia coli can be obtained by amplifying from the chromosomal DNA of Escherichia coli by PCR using primers prepared based on a known base sequence.

The amino acid sequence at positions 15 to 19 of the mutant NAGS of the present invention is any of SEQ ID NOs: 1 to 4.
The known mutant NAGS of Escherichia coli (position 19
SEQ ID NOs: 5 and 6 show the corresponding amino acid sequences of Tyr to Cys) and wild-type NAGS. Examples of the base sequence encoding each amino acid sequence are shown in SEQ ID NOs: 7 to 12. These sequences are shown in Table 1.

[0015]

[Table 1]

Further, the mutant NAGS of the present invention is capable of releasing feedback inhibition by L-arginine,
That is, as long as it has an activity of catalyzing the reaction of acetylating L-glutamic acid to produce N-acetylglutamic acid, substitution or deletion of one or several amino acids at one or more positions other than the 15th to 19th positions. , Insertion or addition.

Here, "several" differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein. This is because some amino acids have highly analogous amino acids, and such amino acid differences do not significantly affect the three-dimensional structure of the protein. Therefore, the total amino acid sequence constituting NAGS is 30 to
It may have a homology of 50% or more, preferably 50 to 70% or more, more preferably 80% or more, and may have NAGS activity of the fbr trait.

In the present invention, the amino acid sequence corresponding to positions 15 to 19 refers to the wild type of Escherichia coli.
A sequence corresponding to the amino acid sequence at positions 15 to 19 in the amino acid sequence of NAGS. Deletion, insertion or addition of an amino acid that does not affect the activity as described above may shift the position of the amino acid residue. For example, if one amino acid residue is inserted at the N-terminal, the amino acid residue at position 15 is originally at position 16, and an amino acid residue corresponding to such an amino acid residue at position 15 is defined in the present invention. Is 1
It is referred to as the amino acid residue at position 5.

A DNA encoding a protein substantially identical to the mutant NAGS as described above may be modified such that amino acid residues at specific sites contain substitutions, deletions, insertions or additions, for example, by site-directed mutagenesis. By modifying the base sequence. In addition, the modified DN as described above
A can also be obtained by a conventionally known mutation treatment. As the mutation treatment, DNA containing the mutant argA gene
In vitro with hydroxylamine or the like, and a microorganism having the mutant argA gene, for example, a bacterium belonging to the genus Escherichia, is irradiated with ultraviolet light or N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrite. A method of treating with a mutagen that is usually used for mutagenesis is exemplified.

In addition, substitution, deletion, insertion or addition of a base as described above may be a mutation (mutant or mutation) that occurs naturally, for example, based on individual differences in the genus, species, or strain of a microorganism that carries NAGS. variants).

By expressing the DNA having the above mutation in an appropriate cell and examining the NAGS activity of the expression product, a DNA encoding a protein substantially identical to the mutant NAGS can be obtained.

Further, a cell having the mutant NAGS is subjected to mutation treatment, and a DNA having a known argA gene sequence is obtained from the cell.
Alternatively, by hybridizing with a probe that can be prepared from the same gene sequence under stringent conditions, and by isolating a DNA encoding a protein having NAGS activity, a protein substantially identical to the mutant NAGS can be obtained. An encoding DNA is obtained.

The term "stringent conditions" as used herein refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to quantify these conditions clearly, as an example, DNAs having high homology, for example, DNAs having homology of 50% or more hybridize to each other, and DNAs having lower homology 1 × SSC at 60 ° C., preferably 65 ° C., which is a condition under which they do not hybridize with each other, or a washing condition for ordinary Southern hybridization.
0.1% SDS, preferably 0.1 × SSC, 0.1
Conditions for hybridization at a salt concentration corresponding to% SDS are exemplified.

Some of the genes that hybridize under such conditions include those in which a stop codon is generated in the middle and those whose activity is reduced or eliminated due to mutation of the active center. It can be easily removed by connecting to a vector and examining NAGS activity.

<2> Escherichia bacterium of the present invention The Escherichia bacterium of the present invention is a bacterium of the genus Escherichia into which the mutant argA gene has been introduced. Specific examples of Escherichia bacteria include Escherichia coli. The mutant argA gene can be introduced by transforming a Escherichia bacterium with a recombinant DNA obtained by ligating a DNA fragment containing the gene to a vector that functions in the bacterium of the genus Escherichia. Alternatively, the mutant argA gene can be introduced by replacing the argA gene on the chromosomal DNA with the mutant argA.

The vectors used for the introduction of the argA gene include plasmid vectors such as pBR322, pMW118 and pUC19.
Phage vectors such as λ1059, λBF101, M13mp9, Mu, T
and transposons such as n10 and Tn5.

The introduction of DNA into Escherichia bacteria is
The method of DAMorrison (Methods in Enzymology 68, 326
(1979)) or by treating recipient cells with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A.,
J. Mol. Biol., 53, 159 (1970)).

When a mutant argA gene is introduced into a bacterium belonging to the genus Escherichia having L-arginine-producing ability as described above, the amount of L-arginine produced can be increased. In addition, L-arginine-producing ability may be imparted to a bacterium belonging to the genus Escherichia carrying the mutant argA gene.

As Escherichia coli having L-arginine-producing ability, Escherichia coli 237 strain (VKPM B-
7925). The shares became Russia on April 10, 2000.
n National Collection of Industrial Microorganisms
(VKPM), deposited under the number VKPM B-7925, May 1, 2001
It was transferred to an international deposit under the Budapest Treaty on August 8.

<3> Method for producing L-arginine As described above, the mutant argA gene was introduced,
Escherichia bacteria having arginine-producing ability is cultured in a suitable medium, L-arginine is produced and accumulated in the medium, and L-arginine is collected from the medium, whereby L-arginine can be efficiently produced. it can.

In the production method of the present invention, the cultivation of Escherichia bacteria and the collection and purification of L-arginine from the culture solution can be carried out in the same manner as the conventional production method of L-arginine by fermentation using microorganisms. Good. As the medium used for the culture, a synthetic medium or a natural medium may be used as long as it contains a carbon source, a nitrogen source, and an inorganic substance, and if necessary, contains an appropriate amount of a nutrient required for the growth of the used strain. Examples of the carbon source include various carbohydrates such as glucose and sucrose, and various organic acids. Further, alcohols such as ethanol and glycerol can be used depending on the assimilation property of the microorganism used. Examples of the nitrogen source include ammonia, various ammonium salts such as ammonium sulfate, amines and other nitrogen compounds, and natural nitrogen sources such as peptone, soybean hydrolyzate, and fermentation cell decomposed product. As inorganic substances, monopotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate,
Manganese sulfate, calcium carbonate and the like are used.

The cultivation is preferably carried out under aerobic conditions such as shaking culture and aeration / agitation culture.
C., preferably in the range of 30 to 38.degree. Medium p
H is usually in the range of 5 to 9, and preferably in the range of 6.5 to 7.2. The pH of the medium can be adjusted with ammonia, calcium carbonate, various acids, various bases, buffers and the like. Usually, L-arginine accumulates in the culture solution by culturing for 1 to 3 days.

After completion of the culture, solids such as cells are removed from the culture by centrifugation or membrane separation, and L-arginine can be collected and purified by ion exchange, concentration, crystallization, etc. .

[0034]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples.

[0035]

Example 1 <1> Mutagenesis with Randomized Fragment A BamHI-SalI chromosomal DNA fragment (2.02 kbp) having a wild-type argA gene was cloned into plasmid pUC19 to obtain plasmid pUC19-ArgA. Pyrobest used for PCR amplification
^TM DNA polymerase was obtained from Takara Shuzo Co., Ltd. (Japan) and used under the conditions recommended by the company.

To create a pool of mutant argA genes,
As a first step, a fragment of the argA gene encoding the sequence from position 20 to the end of NAGS was amplified (FIG. 1). Using plasmid pUC19-ArgA as a template, sense primer P
1: 5'-CGAGGGATTCCGNNNNNNNNNNNNNNATCAATACCCACCGGG-3 '
(SEQ ID NO: 13) was designed based on the base sequence of argA, and a standard M13 reverse primer was used as antisense primer P2. Immobilized 16 of primer P1
The nucleotide 3 'terminal sequence is homologous to the sequence downstream from the Tyr-19 codon of the argA gene, and the fixed 13 nucleotide 5' terminal sequence is homologous to the upstream sequence of the His-15 codon. The homology of the 3 'end of P1 to the argA sequence was used to synthesize a 1.75 kbp DNA fragment by 20 PCR cycles.

Using 100 ng of pUC19-ArgA as a template, a PCR solution (50 μm) containing each of two primers (40 pmol)
l). 20 cycles of PCR (0.6 min at 94 ° C,
0.5 minutes at 55 ° C., 2 minutes at 72 ° C.)
Thermal cycler (Perkin-Elmer Co., Foster City,
Calif.).

In the second stage, eight cycles (one cycle at 94 ° C.)
For 1 minute at 37 ° C. and 0.5 minutes at 72 ° C.). Here, the (−) strand of the amplified fragment functions as a “primer” to extend it to obtain the entire gene sequence.

In the third step, 10 μl of the reaction mixture was added to a sense primer P3: 5′-TGCCATGGTAAAGGAACGTAAAACC-3 ′ (SEQ ID NO: 1) which is homologous to the 5 ′ terminal sequence of the argA gene.
4) and a new reaction mixture (40 μl) containing 100 pmol of the antisense primer P2, and further 10 cycles (0.5 minutes at 94 ° C., 0.5 minutes at 52 ° C., 2 minutes at 72 ° C.) were performed.

A 1.78 kbp DNA fragment encoding a pool of mutant forms of the full-length argA gene was purified by agarose gel electrophoresis, digested with NcoI (the site containing the start ATG codon of the argA gene) and HindIII, and digested with NcoI and It was ligated with the pKK2333-2 vector (Pharmacia, Sweden) digested with HindIII.

About 150 ng of the ligated DNA was used in each of the following experiments. Approximately 2000 recombinant clones were obtained for the transformation of E. coli recipient cells.

<2> Isolation of novel mutant argA and NAGS
Of Amino Acid Substitution on Catalytic Properties of Plasmid Plasmid Vector pKK233-2 (Pharmacia, Sweden)
Was used for cloning and expression of the mutant argA gene. E. coli receiving strain is TG1 (supE hsdD5 thiD (lac ^- p
roAB) F '[traD36 proAB ⁺ lacZDM15]) (J. Sambrook et
al., Molecular Cloning, 1989). Recombinant plasmid pKK-argA-random (has mutant argA gene)
Selection of TG1 cells carrying the set was performed on LB agar plates. Delayed cell growth was observed in some mutant clones, suggesting that this effect was correlated with production of active fbr NAGS mutant enzyme. The plasmid was purified from several clones, and the DNA of the 5 'fragment of the mutant argA gene was
The sequence was determined by the dideoxy chain termination method (Table 2).

[0043] To measure the NAGS activity, arginine auxotroph mutant E. coli B3083 strain (argA ^-, metB ^-) a,
It was transformed with the above plasmid. Enzyme in soluble fraction obtained from sonicated recombinant cells,
Partially purified by ammonium sulfate precipitation and assayed as described below. Plasmid pKK-argA-r11 (3390
nmol / min × mg), pKK-argA-r12 (1220 nmol / min × mg) and pKK-argA-r13 (3120 nmol / min × mg)
The NAGS activity was much higher than the NAGS activity of the strain carrying pKK-argA-r4 (300 nmol / min × mg). The final plasmid was Rajagopal, BS et al. (Rajagopal, BS et al., A
ppl.Environ.Microbiol., 1998, v.64, No.5, p.1805
-1811) retains a mutant argA gene with the same substitution (Y19C) as described as being the most active variant of the argA gene with a single substitution.

The plasmid pKK-argA (wt) (wild type ar
gA) also shows the activity of NAGS in strains carrying pKK-argA-r
11, lower than in the case of pKK-argA-r12 and pKK-argA-r13. The activity levels of the mutant enzymes were almost the same in the presence of 10 mM arginine, whereas the wild-type enzyme was clearly inhibited by arginine (less than 1/10). These results
The peptide fragment of the amino acid residues at positions 15 to 19 is L
-Suggests that arginine plays a role in the catalytic ability of mutant NAGS as well as feedback inhibition of NAGS.

(Enzyme assay) Acetyl coenzyme A
And all compounds used were Sigma Chemical Co.,
Purchased from St. Louis, Mo. To measure NAGS activity, cells containing the recombinant plasmid E. coli B3083 (arg
A ^-, metB ^-) were cultured to late exponential phase in M9 medium (5 ml), and washed with 0.14 M NaCl solution, containing 100 mM KCl in 2ml
Resuspended in 40 mM potassium phosphate buffer (pH 7.0). Cells were sonicated and centrifuged. The fractions containing NAGS are precipitated with 5 volumes of saturated (NH ₄ ) ₂ SO ₄ and the precipitate is washed with ₄ mM containing 100 mM KCl and 30% (vol / vol) glycerol.
It was dissolved in 2 ml of 0 mM potassium phosphate buffer (pH 7.0). NA
The GS solution was mixed with 0.1 ml of the reaction mixture (100 mM Tris-HCl (pH 8.
5), 35 mM KCl, 20 mM L-glutamic acid, 1.2 mM acetyl coenzyme A) and the reaction mixture was incubated at 37 ° C. for 10 minutes.

The reaction was stopped by adding 0.3 ml of ethanol and the reaction mixture was centrifuged. 0.95ml 0.24
A solution of mM DTNB (5,5-dithio-bis-2-nitrobenzoate) was added to the supernatant and the mixture was incubated for 15 minutes. NA
GS activity was analyzed by monitoring the absorbance at 412 nm.

<3> Novel argA mutation B16-4 (pro^- arg
D^-) Isolation by selection in cells argA mutant (pKK-argA-rando) in E. coli B16-4 strain
The selection of m) was performed by the method described above. Cold
The recombinant clone from the top plate was replaced with L-arginine.
Suspend in M9 medium containing, and culture until stationary phase.
Suspend a portion in a fresh medium and repeat the same culture procedure four times.
I returned. After these, a portion of the culture was replaced with 5 mg / ml L
M9 cold with arginine and 100 μg of ampicillin
Plated on natural medium. Plunge from several clones
The plasmid was purified and the 5 'fragment of the mutant argA gene was identified.
And sequence the level of activity of the mutant NAGS
Assay was performed as described above. 60% of variants are enzyme variants
The sequence -Gly-Trp-Pro-Cys-Val- (SEQ ID NO:
4) and weak (about 10 nmol / min × mg), but fbr NAG
It had S activity. Obviously, this mutant protein
Is pro with the selection criteria used ^- argD^-For cells to grow
Provides optimal levels of NAGS activity. Therefore,
Depending on the selection conditions, the activity of the resulting mutant NAGS is determined.
It is thought that.

[0048]

[Table 2]

<4> Production of L-arginine using mutant argA gene Recombinant plasmids pKKArgA-r4, pKKArgA-r11, pKKArgA-r
12, pKKArgA-r13 and pKKArgA-r32 with BamHI and SalI
The fragment containing the mutant argA gene under the trc promoter was subcloned into the low copy promoter pMW119 (Nippon Gene, Tokyo). The resulting plasmids were named pMADS4, pMADS11, pMADS12, pMADS13 and pMADS32, respectively. These plasmids are
-Escherichia coli 237 strain (V
KPM B-7925). The production of L-arginine (Arg) and citrulline (Cit) of the transformants is shown in Table 3. Most of the production strains carrying the novel mutant NAGS showed higher productivity for L-arginine and citrulline than the recipient strain or the known Tyr19Cys mutant NAGS (pMADS4).

[0050]

[Table 3]

(Culture conditions issued in a test tube)
In 1 L of tap water, 60 g / L glucose, 25 g / L ammonium sulfate, 2 g / L KH ₂ PO ₄ , 1 g / L MgSO ₄ , 0.1 mg / L thiamine, 5 g / L yeast extract ( Difco), 25
g / L Contains precipitated calcium carbonate (pH 7.2). Glucose and precipitated calcium carbonate were sterilized separately. 2 ml of the medium was placed in a test tube, and one loopful of the test microorganism was inoculated and cultured at 32 ° C. for 3 days with shaking.

[0052]

According to the present invention, L of Escherichia bacteria
-The ability to produce arginine can be improved.

[0053]

[Sequence List] SEQUENCE LISTING <110> Ajinomoto Co., Inc. <120> New mutant N-acetylglutamate synthase and method for L-arginine prod uction <130> P-7412F <140> <141> 2001-06-14 <150> RU 2000 116 481 <151> 2000-06-28 <150> RU 2001 112 869 <151> 2001-05-15 <160> 14 <170> PatentIn Ver. 2.0

<210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: partial amino acid sequence encoded by random sequence <400> 1 Val Val Trp Arg Ala 15

<210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: partial amino acid sequence encoded by random sequence <400> 2 Leu Phe Gly Leu His 1 5

<210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: partial amino acid sequence encoded by random sequence <400> 3 Ser Ala Ala Ser Arg 15

<210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: partial amino acid sequence encoded by random sequence <400> 4 Gly Trp Pro Cys Val 15

<210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: partial amino acid sequence encoded by random sequence <400> 5 His Ser Val Pro Cys 15

<210> 6 <211> 5 <212> PRT <213> Escherichia coli <400> 6 His Ser Val Pro Tyr 15

<210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: random sequence <400> 7 gtagtatggc gggca 15

<210> 8 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: random sequence <400> 8 ttgttcggat tgcac 15

<210> 9 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: random sequence <400> 9 tcggcggcgt ccaga 15

<210> 10 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: random sequence <400> 10 gggtggccat gcgtg 15

<210> 11 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: random sequence <400> 11 cattcggttc cctgt 15

<210> 12 <211> 15 <212> DNA <213> Escherichia coli <400> 12 cattcggttc cctat 15

[0066] <210> 13 <211> 42 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: primer containing random sequence <220><221> misc _ feature <222> (13) .. (26) <223> n = a or g or c or t <400> 13 cgagggattc cgnnnnnnnn nnnnnnatca atacccaccg gg 42

<210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 14 tgccatggta aaggaacgta aaacc 25

[Brief description of the drawings]

FIG. 1 shows a scheme for constructing a pool of mutant argA genes.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:19) C12N 15/00 ZNAA (72) Inventor Sergey Vasilievich Smirnov Russia, 121552, Moscow City, Yaltsevskaya Street No. 29, Building No. 2, Room 94 Moscow City, Rodoch Naya Street, No. 31, Building No. 5, Room 77 (72) Inventor Tatjana Vicrovna Leonova, Russian Federation, 123481, Moscow City, Suva Boddy Street No. 81, Building No. 5, Room 852 (72) Inventor Mi Il Markowich Gshachiner Russian Federation, 113646, Moscow, Moscow, Severnoe Certanovo 4, Building 403, Room 217 F-term (reference) 4B024 AA01 AA03 AA05 BA07 BA71 CA04 DA06 EA04 GA11 HA03 4B050 CC01 CC03 DD02 LL05 4B019 AE24 CA02 DA02 DA16 4B065 AA26X AA26Y AB01 BA01 CA17 CA27

Claims (6)

[Claims] An amino acid sequence corresponding to positions 15 to 19 of a wild-type N-acetylglutamate synthase has an amino acid sequence substituted with any one of SEQ ID NOs: 1 to 4, and A mutant N-acetylglutamate synthetase in which feedback inhibition by arginine has been released. 2. The mutant N-acetylglutamate synthetase according to claim 1, wherein the wild-type N-acetylglutamate synthase is Escherichia coli N-acetylglutamate synthase. 3. A method comprising one or more amino acid substitutions, deletions, insertions or additions at one or more positions other than positions 15 to 19, and wherein feedback inhibition by L-arginine has been released. Item 3. The mutant N-acetylglutamic acid synthase according to Item 1 or 2. 4. A D encoding the mutant N-acetylglutamate synthetase according to any one of claims 1 to 3.
NA. 5. Transformation with the DNA according to claim 4,
A bacterium belonging to the genus Escherichia having an ability to produce L-arginine. 6. The Escherichia bacterium according to claim 5, which is cultured in a medium, and L-arginine is produced and accumulated in the medium.
A method for producing L-arginine, comprising collecting L-arginine from the medium.